Rabbit anti phospho s6 s235 236

The following primary antibodies were obtained from Cell Signaling Technology (catalog number, dilution in OBB): rabbit anti-phospho-AKT T308 (13038, 1:400), rabbit anti-phospho-AKT S473 (4060, 1:200), rabbit anti-phospho-Chk1 S317 (12302, 1:800), rabbit anti-phospho-Chk2 T68 (2661, 1:100), rabbit anti-phospho-4E-BP1 T37/46 (2855, 1:200), rabbit anti-phospho-S6 S235/236 (4858, 1:1000), rabbit anti-phospho-H2A.X S139 (“γH2A.X”) (9718, 1:400), rabbit anti-phospho-Histone H3 S10 (3377, 1:800), mouse anti-β-tubulin (86298, 1:400), rabbit anti-β-tubulin (2128, 1:400), mouse anti-BrdU (Bu20a) (5292, 1:200), rabbit anti-phospho-GSK3B S9 (5558, 1:400), rabbit anti-FOXO3 (2497, 1:200), rabbit anti-p21 (2947, 1:400), rabbit anti-p27 (3686, 1:400), and rabbit anti-phospho-Rb S807/811 (8516, 1:800). The following primary antibodies were obtained from Abcam (catalog number, dilution in OBB): rabbit anti-cyclin D1 (ab134175, 1:100), rabbit anti-geminin (ab195047, 1:100), and rabbit anti-cyclin A2 (ab181591, 1:500). Rabbit anti-phospho-RPA S4/8 antibodies were obtained from Bethyl (A300-245A, 1:400), rabbit anti-γ-tubulin antibodies from Sigma (T5192, 1:400), and Human Antibody against Centromere (CREST) antibodies from ImmunoVision (HCT-0100, 1:1000).

The coronal brain slices of 50μm or 150μm thickness were cut using a vibratome (Leica VT1000 S). These slices were permeabilized with PBS containing 0.4% Triton X-(PBST) for 30 min and blocked with PBS containing 10% donor horse serum (DHS) for 1 h at room temperature. Sections were incubated with primary antibodies (Chicken anti-GFP, 1:3000, abcam#13970; Rabbit anti-mCherry, 1:3000, abcam#167453; Mouse anti-mCherry, 1:3000, TaKaRa#632543; Rabbit anti-phospho-S6 S235/236, 1:200, Cell Signaling#4858; Rabbit anti-phospho-AKT-Thr308, 1:100, cell signaling#13038; Rabbit anti-phospho-AKT-Ser473, 1:100, Cell Signaling#9271) for 48 h at 4°C in PBS with 2.5% DHS. After three 15 min washes with PBST, sections were incubated for 48 h at 4°C with secondary antibodies (Alexa Fluor 488 conjugated goat anti-chicken IgG, Jackson ImmunoResearch Labs#103-545-155; Cy3 conjugated donkey anti-rabbit IgG, Jackson ImmunoResearch Labs#711-165-152; Cy3 conjugated donkey anti-mouse IgG, Jackson ImmunoResearch Labs#715-165-150; Alexa Fluor 647 conjugated goat anti-rabbit IgG, Jackson ImmunoResearch Labs#111-605-144) at a concentration of 1:200 in PBS containing 2% DHS. After three 15 min washes with PBST, sections were mounted in Vectashield (H-1000, Vector Laboratories) on glass slides.